Dear all,

I am trying to establish a protocol to assess the in-vitro lipoxygenase inhibition using soybean lipoxygenase (type V). During the protocol, first, we mixed 10 µL of samples with 45 µL of 1000 U/mL enzyme (the enzyme was dissolved in 50 mM Tris buffer, 7.4 pH) for 5 min at 25-celsius degrees. Then the samples were mixed with 45 µL of 2 mM linoleic acid for another 20 min at 25-celsius degrees. Then the samples were mixed with 100 µL of FOX reagent for another 20 min at 25-celsius degrees and absorbance were measured at 560 nm.

Composition of FOX reagent

Sulfuric acid- 30 mM

Xylene orange- 100 µM

Iron (II) sulphate heptahydrate- 100 µM

(This mixture was orange in colour)

Then 1 part of this mixture was mixed with 9 parts of (1:9) of methanol and the colour was changed into purplish.

Linoleic acid was prepared as follows. A 25 mM stock solution was prepared by adding 155 μL (140 mg) of linoleic acid and 257 μL (280 mg) of Tween 20 to 5 mL of water. Then, 0.6 ml 1N NaOH was added and volume up to 20 ml. Finally, the solution was divided into 1 mL aliquots stored at -20-celsius degrees.

I have used 100 µM quercetin as the positive control. Unfortunately, I couldn’t observe any inhibitory effect compared to the control group. I have tried with the lowered concentrations of enzyme and linoleic acid but still, the results are the same.

Please share your experience and help me to establish this protocol.